A large number of low molecular compounds used as pharmaceutical agents are still unclear in regard to their mechanism of action despite their distinct pharmacological actions. In general, most of the pharmaceutical agents act upon specific proteins in vivo and alter functions of the proteins, and induce pharmacological actions as a result. In the agents whose mechanisms of action are unclear, proteins as their targets have not been identified. In recent years, as a result of advances in the elucidation of signal transduction system in vivo at the molecular level, a large number of specific protein molecules necessary for inducing specific pharmacological actions have been identified. As a result, development of the agents so-called molecule targeting agents which target at such specific protein molecules is in progress recently, and their ratio is rapidly increasing. In the case of a compound whose target protein is evident, its in vivo functional mechanism is clear, and structure of the compound can be modified with the use of its strength to bind to said protein, or a change in the enzyme activity possessed by the protein, as the index. Thus, it is easy to carry out studies with the aim of improving pharmacokinetics including absorption and degradation, as well as pharmacological activity, so that it is markedly advantageous in developing agents. In the case of a compound whose target protein is unclear to the contrary, it is not easy to attempt improvement of its chemical structure for the purpose of improving the activity, even when distinct pharmacological action is found (cf. Non-patent Reference 1).
In addition, though there are differences in degree, pharmaceutical agents generally have both desirable pharmacological actions (principal effects) and undesirable pharmacological actions (adverse side effects). Even in the case of molecule targeting agents whose target proteins carry the principal effects and are already put on the market, there are many cases in which information on the target proteins concerned in the adverse side effects is scanty, and this causes a problem requiring time and cost for studying improvement for avoidance of the adverse side effects (cf. Non-patent Reference 1).
In fact, there are a large number of pharmaceutical agents whose significant pharmacological actions are known but their target proteins are unclear. As representative examples, biguanide which has been used for a long time as an agent for treating diabetes (cf. Non-patent Reference 2) and thalidomide in which its presence has been reconsidered in view of its drastic therapeutic effect on multiple myeloma may be cited (cf. Non-patent Reference 3). Though biguanide has a significant hypoglycemic action, and thalidomide a significant angiogenesis inhibitory action, direct target protein for each of these agents in vivo has not been identified. Thus, in spite of the useful pharmacological actions possessed by these agents, it was difficult to carry out improvement studies for enhancing the effects. In addition to this, serious adverse side effects such as lactic acidosis by biguanide (cf. Non-patent Reference 4) and teratogenicity by thalidomide (cf. Non-patent Reference 3) are known, but studies for avoiding these problems have not been advanced, because their targets are unclear. Thus, identification of the target proteins of these agents is in demand.
Conventionally, a method in which a protein which directly binds to a low molecular compound is detected and separated by physical and/or chemical means was a general means for identifying a target protein upon which said compound acts. For example, a method is known in which a part of the structure of a compound is modified and bind to high molecular weight affinity beads, and a target protein bound to the compound is separated and purified by gravity or the like physical force. Also, a method is carried out in which a tag to be used as a label is attached to a part of the structure of a compound and the target protein bound to said compound is chemically detected (cf. Non-patent Reference 5). In recent years, attempts have also been made to screen and identify, from a cDNA library, a gene fragment coding for a protein which binds to the compound of interest, by a yeast two hybrid method (cf. Non-patent Reference 6), a phage display method (cf. Non-patent Reference 7) and the like molecular biological techniques.
However, in spite of the aforementioned attempts by various methods, a case in which a target protein of an agent was actually identified from the studies in this field so far is not many. The reasons for the low frequency of success include that it is necessary to modify a part of the structure of a compound because beads or a tag is bound to the compound to be used as the probe in every case of the aforementioned methods, so that it is unavoidable to screen for a protein which binds to an artificial structure different from the original compound (cf. Non-patent References 1 and 5). That is, this becomes a reason of mistakenly identifying, as the target protein, a nonspecific protein which binds to a tag, beads, a complex thereof with a compound, or the like artificial substance which is different from the agent having original pharmacological activity. In addition, though it is essential, for the purpose of finding the true target protein, to apply modification of the structure of a compound to a region which does not exert influence upon the pharmacological action of said compound, agents and compounds having unclear targets are generally poor in information on the correlation between their structures and pharmacological activities, so that there are many cases in which compounds modified at optional regions have to be used. Because of this, there is a high frequency of selecting a compound which lost its original pharmacological activity as the probe. Essentially, it is desirable to verify firstly that said compound to which a tag or bead is added by modification is still keeping its original pharmacological activity and then use it as the probe, but since cell membrane permeability, stability and the like various parameters exert influences, it is not easy to judge the presence or absence of the pharmacological activity. Also, since modification of the structure of compounds requires time, cost and special techniques, these cause the aforementioned methods to hardly become a general purpose studying means.
On the other hand, it is possible to verify binding of a specified protein to a compound labeled by replacing an element in the molecule of a compound with a radioisotope (its structure is the same as the before labeling), but since it is not a fixable modification, it is not easy to screen the target protein from a large number of proteins. In addition, this method has a disadvantage in that the compound becomes unstable by the labeling and the cost runs up.
As another reason of the low success ratio of compound target screening by the conventional methods, a point can be exemplified that since each of the aforementioned methods carries out detection and separation of a target making use of the direct binding of a compound with a protein as the index, it is difficult to achieve target finding when the binding affinity between the compound and the target protein is low. Actually, in each of the only few cases of succeeding in finding a target by the aforementioned methods, the binding affinity between the compound and the protein is high (cf. Non-patent Reference 4). However, the degree of pharmacological activity of a compound and its binding affinity for a target protein do not always have a correlation based on the knowledge so far obtained. Rather, it is considered that strong binding of a compound to target protein may not be necessary for the induction of pharmacological action excluding irreversible inhibition (cf. Non-patent Reference 8). Based on the above problems, concern has been directed toward a method for identifying target proteins of agents, which were not able to be found by the conventional methods.
Molecular chaperone is a group of proteins which assist structure formation of protein, such as folding or denaturation (unfolding) of a protein molecule, multimer formation and the like (cf. Non-patent Reference 9). It is known now that a large number of molecules generally referred to as heat shock protein, in which its expression is accelerated by heat stimulation, act as chaperone. Among the molecular chaperones, a group of molecules generally referred to as Hsp60 family are particularly called “chaperonin” as a typical molecular chaperone.
These molecular chaperones represented by the heat shock protein interact with unstable proteins before completion of their tertiary structures in their translation process and keep them stably, and also have the action to maintain and control the protein structure such that influences upon the function of intracellular protein are not caused accompanied by an environmental change and to accelerate ubiquitination and subsequent degradation of substrate which became an abnormal state (cf. Non-patent Reference 10).
Thus, the chaperone has a property as a functional molecule which recognizes non-natural structure of a protein molecule as the substrate.
On the other hand, a screening method for identifying the ligand of already known target protein has been reported, which uses molecular chaperon for the determination of the degree of folded state and unfolded state of the target protein in the presence or absence of a ligand candidate (cf. Non-patent References 1 to 6).
Insulin is secreted from the 13 cell of pancreatic islets of Langerhans and reduces blood sugar level by acting mainly upon muscles, the liver and fat to store and consume blood sugar through its intake into cells. Diabetes is induced by the insufficient action of this insulin, and there are two types in its patients, namely, type I having a disorder in the production or secretion of insulin, and type II in which acceleration of glucose metabolism by insulin becomes difficult to occur. Though the blood sugar level becomes higher than that of healthy people in both of these patients, blood insulin becomes absolutely scarce in type I, while insulin resistance in which intake or consumption of blood sugar by cells is not accelerated in spite of the presence of insulin is generated in type II. The type II diabetes is a so-called life style-related disease which is induced by overeating, less exercise, stress and the like causes in addition to hereditary basic factor. These days, this type II patient is rapidly increasing in advanced nations accompanied by the increase of caloric intake, and it occupies 95% of diabetes patients in Japan. Thus, the necessity of not only a simple hypoglycemic agent but also treatment of type II diabetes for accelerating glucose metabolism through the improvement of insulin resistance is increasing as agents for treating diabetes.
Currently, insulin injections are prescribed for the treatment of type I diabetes patients. On the other hand, as the hypoglycemic agent prescribed for type II diabetes patients, a sulfonylurea system hypoglycemic agent (SU agent) which accelerates secretion of insulin by acting upon β cells of the pancreas and an α-glucosidase inhibitor which delays digestion absorption of glucose are known, in addition to the insulin injections. Though these improve insulin resistance indirectly, a thiazolidine derivative has been used in recent years as an agent which more directly improves insulin resistance. Its action is to accelerate intake of glucose into cells and use of glucose in the cells. It has been shown that this thiazolidine derivative acts as an agonist of peroxisome proliferator activated receptor gamma (PPAR γ) (cf. Non-patent Reference 11). However, it is known that the thiazolidine derivative not only improves insulin resistance but also has adverse side effects of inducing fat accumulation and edema (cf. Non-patent Reference 12). Since this induction of edema is a serious adverse side effect which results in cardiac hypertrophy, more useful new drug target molecule instead of PPAR γ is in demand for the improvement of insulin resistance. As a leading agent which produces a glucose metabolism improving action other than these, a hypoglycemia agent biguanide which has been used for a long time is known (cf. Non-patent Reference 13). The biguanide agent has been reported to have actions to enhance glucose metabolism by anaerobic glycolytic action, suppression of gluconeogenesis, suppression of appetite and suppression of intestinal absorption of glucose, and as a result, biguanide improves insulin sensitivity in the liver and muscles. Since biguanide does not act upon the pancreas and does not increase secretion of insulin, it has a characteristic in that it does not cause obesity and hardly cause hypoglycemia. The action of biguanide does not include undesirable actions possessed by the aforementioned thiazolidine derivative and insulin preparations, and there are many cases in which it is prescribed in combination with the aforementioned other hypoglycemic agents in reality. Combined with the recent year's reconsideration on its strong pharmacological action, the biguanide agent now holds its position net to the thiazolidine derivative as insulin resistance improving agent. But on the other hand, it is known that biguanide agent has an adverse side effect of causing lactic acidosis by increasing accumulation of lactic acid (cf. Non-patent Reference 14). In spite of the very old history of biguanide as an agent, a distinct target protein, like the case of PPARγ of the thiazolidine derivative, has not been identified yet. Since information on the structural activity correlation regarding biguanide agents and the target protein has not been obtained, not only a dissociate study on adverse side effect such as improvement of lactic acidosis but also an improvement study aimed at increasing hypoglycemia as the principal effect has been difficult to carry out up to the present. ATP5B protein is the β subunit of F1F0-ATP synthase, which is encoded on the genome and perform its action after transferred to mitochondria (cf. Non-patent References 15 and 16). Also, regarding the existing amounts of ATP5B, it has been reported that both of the amounts of its gene expression and protein amount are lowered in muscles of type II diabetes patients in comparison with those of healthy people (cf. Non-patent References 17 and 18 and Patent Reference 7). In addition, it has been reported that phosphorylation level of ATP5B in muscles of diabetes patients and fasting blood sugar level take inverse correlation (cf. Non-patent Reference 18 and Patent Reference 7), and those (e.g., a nucleic acid fragment) which control expression of ATP5B, a polypeptide, an antibody, a polynucleotide or a compound which binds to a polypeptide, and the like can be agents for treating diabetes-associated diseases (cf. Patent Reference 7). There is a report which discloses various polypeptides (3025 substances) included in human heart mitochondrial proteome including ATP5B, and describes that these are related to the screening for an agent for treating diseases (including diabetes) associated with mitochondrial functions (cf. Patent Reference 8). However, there are no reports stating that ATP5B protein binds to biguanide.    Patent Reference 1: U.S. Pat. No. 5,585,277    Patent Reference 2: U.S. Pat. No. 5,679,582    Patent Reference 3: US Patent Application Publication No. 2002/055123    Patent Reference 4: US Patent Application Publication No. 2004/191835    Patent Reference 5: Japanese Patent No. 2952848    Patent Reference 6: European Patent No. 0770876    Patent Reference 7: International Publication No. 03/020963    Patent Reference 8: International Publication No. 03/087768    Non-patent Reference 1: “The Journal of Antibiotics” H. Hatori et al., 2004 vol. 57 no. 7 p. 456-461    Non-patent Reference 2: “Nippon Rinsho (Japan Clinics)” Y. Yamacaki et al., 2002 vol. 60 no. 9 p. 389-92    Non-patent Reference 3: “Drug Discovery Today” Teo S K et al., 2005 vol. 15 no. 10(2) p. 107-114    Non-patent Reference 4: “Drugs” Lalau J D et al., 1999 vol. 58 no. 1 p. 55-60/75-82    Non-patent Reference 5: “Nature Biotechnology” (England) 2000, N. Shimizu et al., vol. 18, p. 877-881    Non-patent Reference 6: “Biochemical Pharmacology” 2002, D. Henthorn et al., vol. 63 no. 9 p. 1619-1628    Non-patent Reference 7: “Chemistry & Biology” Sche P P et al., vol. 6 no. 10: p. 707-716. PMID: 10508685    Non-patent Reference 8: “Biochemistry (OUTLINES OF BIOCHEMISTRY)” 1987, Eric E. CONN et al.    Non-patent Reference 9: “Pharmacology & Therapeutics” 2004, A. Sreedhar et al., vol. 101 no. 3 p. 227-257    Non-patent Reference 10: “Nature” 1992, Gething M J, Sambrook J. et al., vol. 355 no. 6355: p. 33-45    Non-patent Reference 11: “The Journal of Biological Chemistry”, (USA), 1995, vol. 270, p. 12953-12956    Non-patent Reference 12: “Diabetes Frontier”, (USA), 1999, vol. 10, p. 811-818    Non-patent Reference 13: “Nippon Rinsho (Japan Clinics)” Y. Yamasaki et al., 2002 vol. 60 no. 9 p. 389-92    Non-patent Reference 14: “Drugs” Lalau J D et al., 1999 vol. 58 no. 1 p. 55-60/75-82    Non-patent Reference 15: “Nature” (USA), 1997, vol. 386, p. 299-302    Non-patent Reference 16: “Nature” (USA), 1994, vol. 370 (6491), p. 621-628    Non-patent Reference 17: “Diabetes” 2002, vol. 51, p. 1913-1920    Non-patent Reference 18: “The Journal of Biological Chemistry” 2003, vol. 278, p. 10436-10442